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Syndecan-4 up-regulation in proliferative renal disease is related to microfilament organization¹

SUSAN YUNG^*,2, ANNE WOODS, TAK MAO CHAN, MALCOLM DAVIES^*, JOHN D. WILLIAMS^* and JOHN R. COUCHMAN³

^* Institute of Nephrology, University of Wales College of Medicine, Cardiff, Wales, UK;
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama, 35294, USA; and
Division of Nephrology, Department of Medicine, The University of Hong Kong, Hong Kong SAR, China

³Correspondence: Volker Hall 201, Department of Cell Biology and Cell Adhesion and Matrix Research Center, 1530 3rd Ave. South, University of Alabama at Birmingham, Birmingham AL 35294-0019, USA. E-mail: jrcouchman{at}cellbio.bhs.uab.edu

SPECIFIC AIMS

The purpose of this study was to gain molecular understanding of the changes in cell–extracellular matrix interactions that characterize IgA nephropathy. Syndecan-4, a transmembrane heparan sulfate proteoglycan, has been proposed as an early response gene in vascular injury. We hypothesized that the specialized glomerular vascular bed may exhibit changes in syndecan-4 expression, with concomitant alteration of the actin cytoskeleton.

PRINCIPAL FINDINGS

1. Syndecan-4 mRNA and protein up-regulation in proliferative but not nonproliferative kidney disease
Biopsies from patients diagnosed with IgA nephropathy or nonproliferative thin membrane disease were subjected to semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and indirect immunofluorescence microscopy. In both assays, a marked up-regulation of syndecan-4 mRNA (sixfold) and core protein expression were noted in every case of IgA nephropathy compared with thin membrane disease (Fig. 1 ). Immunofluorescence microscopy of IgA nephropathy biopsies showed abundant syndecan-4 within the glomeruli, probably in mesangial matrix, as well as in the cortical interstitium (Fig. 2 ). Similar changes were not seen with the basement membrane proteoglycan perlecan, previously shown to be a mesangial matrix heparan sulfate proteoglycan.

View larger version (35K): [in this window] [in a new window]   Figure 1. RT-PCR analysis of mRNA for syndecan-4 in renal biopsies obtained from thin membrane nephropathy (lanes 1–5) or IgA nephropathy (lanes 6–11). 10 ng of total RNA from each sample was reverse transcribed into cDNA and amplified for the syndecan-4 gene using PCR. Syndecan-4 is represented by the 520 bp product and the housekeeping gene -actin by the 204 bp product.

View larger version (99K): [in this window] [in a new window]   Figure 2. Representative immunohistochemical staining of syndecan-4 and -actinin in normal kidney (a, b), thin membrane disease (c, d), and IgA nephropathy (e, f). Staining of syndecan-4 in the normal tissue and thin membrane nephropathy is concentrated within the mesangium (a and c, respectively), whereas increased staining is observed in IgA nephropathy both within the mesangium and tubulo-interstitium (e). Similar staining patterns are observed with -actinin (b, d, f). Bar = 30 µm.

2. Increased -actinin staining accompanied that of syndecan-4 in IgA nephropathy biopsies
A parallel increase in the staining for an important actin-associated protein, -actinin was noted in all biopsies from IgA nephropathy patients (Fig. 2) . This was not seen in the biopsies from patients with thin membrane disease. This increase was also noted in both the interstitium and glomeruli, in a pattern almost identical to that of syndecan-4 proteoglycan. Other microfilament-associated proteins, including vinculin and paxillin, showed similar changes in proliferative kidney disease. All these cytoskeletal proteins are found in specialized cell–matrix adhesion organelles, known as focal adhesions or focal contacts. Previous reports indicate that syndecan-4 is also a focal adhesion component and might be required for focal adhesion assembly.

3. Human mesangial cells in primary culture expressed syndecan-4 as a focal adhesion component
Further RT-PCR performed on primary human mesangial cells showed that syndecan-1 and -4 were expressed at the mRNA level, confirmed by indirect immunofluorescence microscopy with core protein-specific antibodies. Glypican-1, an unrelated cell surface heparan sulfate proteoglycan, and perlecan were also detected at the mRNA level. Cell surface proteoglycan expression was not influenced by the presence of serum in the culture medium except in the case of syndecan-1, whose mRNA levels were increased in the presence of 20% FCS. Proteoglycan purification and analysis by enzyme treatments and Western blotting showed that syndecan-4 was expressed as a heparan sulfate proteoglycan by mesangial cells, as shown previously in other cell types. Moreover, syndecan-4 was retained in Triton X-100-resistant preparations of these cells, indicating a linkage to the cytoskeleton and/or extracellular matrix.

Indirect immunofluorescence microscopy revealed that syndecan-4 was a focal adhesion component in primary mesangial cells, where it codistributed with other known components including vinculin, paxillin, selected integrins, and -actinin. Double staining also showed, as required for the definition of focal adhesions, that syndecan-4 was present at the termini of microfilament bundles. The presence of focal adhesions was also confirmed by interference reflection microscopy. The use of detergent-resistant preparations again revealed a close association of -actinin and syndecan-4, since both were specifically retained and codistributed in mesangial cells. They were present in focal adhesions and in a cortical, submembranous sheath of microfilaments characteristic of smooth muscle cells. The use of Triton X-100 treated live cell preparations also showed that other actin-associated proteins such as talin, vinculin, and paxillin were almost completely lost.

CONCLUSIONS

IgA nephropathy is characterized by mesangial proliferation, the local production of cytokines, and excess extracellular matrix deposition within the glomerulus. It remains a disease without satisfactory treatment, and the underlying molecular pathology is poorly understood. Syndecan-4 is a heparan sulfate proteoglycan reported to be an early response gene product in vascular injury, though its distribution and role in kidney diseases has not been investigated before. We hypothesized that altered vascular cell–matrix interactions, which underpin IgA nephropathy, might involve syndecan-4 since it is a regulator of cytoskeletal organization. We found that syndecan-4 was markedly up-regulated at the mRNA and protein level in each biopsy from IgA nephropathy patients, but not in biopsies from thin membrane disease. Immunofluorescence microscopy showed glomerular and interstitial distributions of the proteoglycan along with substantially increased levels of actin-associated proteins, including -actinin. Further work with mesangial cell cultures showed specific association of syndecan-4, a transmembrane receptor that influences integrin-mediated adhesion, with -actinin. These two molecules were selectively retained in detergent-resistant preparations and are present in focal adhesions.

Many anchorage-dependent cells in culture assemble focal adhesions and equivalent in vivo structures include smooth muscle dense bodies and the fibronexus structures (microfilament–matrix complexes) formed by ‘activated’ fibroblasts in wound contraction. Mesangial cells can become activated in the course of proliferative diseases, with the expression of -actin and excess extracellular matrix production. Cytokines such as transforming growth factor-ß (TGF-ß) have often been implicated in these processes. Syndecan-4 is not known to be a TGF-ß-responsive gene, but tumor necrosis factor does up-regulate syndecan-4 synthesis. Syndecan-4 regulates actin cytoskeletal organization, and it therefore seems probable that its expression is an important part of mesangial and fibroblast ‘activation’. Syndecan-4 can also function as a coreceptor for growth factors such as fibroblast growth factors and so might also be involved in signal transduction by heparin binding growth factors. Integrins have been shown to be unchanged in IgA nephropathy. It seems, therefore, that the modulation of integrin function by syndecan-4 may coordinate changes in cytoskeletal architecture, and its levels may be a sensitive marker of the proliferative/fibrotic state of mesenchymal cells. The potential involvement of syndecan-4 in proliferative IgA nephropathy is shown in Fig. 3 .

View larger version (25K): [in this window] [in a new window]   Figure 3. Chart to show the relationship of syndecan-4 expression and cytoskeletal reorganization with the pathogenesis of IgA nephropathy.

Smooth muscle actin cytoskeleton is one factor that can control vascular tone and, when in a highly developed state (as seen here in IgA nephropathy) may contribute to changes in blood pressure in the glomerulus. This in turn can lead to further deleterious changes in glomerular function and progressive disease. Further work must be undertaken to evaluate the signaling processes that emanate from syndecan-4. Work with fibroblasts has shown a direct interaction of syndecan-4 with protein kinase C that also involves inositol phospholipids. Furthermore, our recent and ongoing work shows that there may be a direct association of -actinin with syndecan-4. The potential for cytoskeletal regulation through syndecan-4 therefore may be direct and may involve PKC phosphorylation of cytoskeletal targets, including -actinin.

FOOTNOTES

¹ To read the full text of this article, go to ttp://www.fasebj.org/cgi/doi/10.1096/fj.00-0794fje ; to cite this article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0794fje

² Current address: Division of Nephrology, Department of Medicine, The University of Hong Kong, Hong Kong SAR, China.

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